JP4337644B2 - Manufacturing method for biochemical instruments - Google Patents

Description

The present invention also relates to the manufacture how the biochemical instrument.

  The surface of plastic or glass products has the characteristic that non-specifically adsorbed biological substances such as proteins, but life that makes full use of culture techniques such as plant tissue culture and animal cell culture and immunobiochemical techniques Non-specific adsorption may cause various problems when molded products made of these materials are used in blood test containers for specimens used in scientific fields, specimen storage containers, and containers for storing biological reagents. cause.

For example, in test vessels in the biochemical field, if the sample is adsorbed in the dilution or storage process, the concentration changes, which greatly affects the analysis results. In blood collection containers and sample storage containers, proteins in samples such as blood Is adsorbed in the container, the test value of the protein amount is lower than the actual value or not detected at all, and there is a possibility that the diagnosis is wrong.

  In addition, with the progress of protein structure analysis technology in recent years, a very small amount of protein has become a subject of research, and even in these studies, accurate analysis results cannot be obtained if the protein and peptides constituting the protein are adsorbed to the container. Furthermore, since various organic solvents, surfactants, acids and the like are used in the protein structure analysis step, the containers used are required to have resistance to organic solvents, surfactants and acids.

  As a means for solving such a problem, generally, a method of adding a protein or a surfactant, such as bovine serum albumin, which is easily adsorbed as a contaminant.

  However, in these methods, the effect of preventing adsorption is hardly obtained, and the added substance itself may affect the analysis / evaluation results.

Moreover, a method of modifying the surface of a polystyrene member by circulation of ozone gas is disclosed (for example, see Patent Document 1). However, it is known that polar groups introduced on the polystyrene surface will sink into the resin layer over time due to contact with the gas phase, and the treatment effect will decrease over time. It was.
JP, 10-101820, A There is also known a method of constructing a hydrogel layer by grafting a hydrophilic material such as polyethylene oxide on the surface of a container to reduce adsorption of a biological substance. In this method, it is difficult to uniformly control the length of the graft chain, and further, it is difficult to increase the density of graft chain introduction, so there is a large variation in modification and it is difficult to obtain a sufficient modification effect. Had problems.

  In addition, a method of coating the surface of the container with a hydrophilic material has been conventionally used, but the resistance to organic solvents and surfactants is weak, and the effect cannot be obtained.

  An object of the present invention is to provide a surface modification method for reducing adsorption of a biological substance to the surface of a biochemical instrument and a biochemical instrument manufactured by the method.

Such an object is achieved by the present invention described in the following (1) to (6).
(1) a first step of immersing a biochemical instrument in a water-soluble resin having a first functional group in a side chain to form a coating layer on the surface of the biochemical instrument;
A second step of curing the coating layer,
The method for producing a biochemical instrument, wherein the water-soluble resin having the first functional group in the side chain is represented by the following formula (Ia ) .

(2) The method for producing a biochemical instrument according to (1), wherein a second functional group is previously formed on the surface of the biochemical instrument.
(3) The method for producing a biochemical instrument according to (1) or (2), wherein the biochemical instrument is made of a resin.
( 4 )
The method for producing a biochemical instrument according to any one of (1) to ( 3 ), wherein the second step is to cure the coating layer by light irradiation.
( 5 )
The method for producing a biochemical instrument according to any one of (1) to ( 3 ), wherein the second step is to cure the coating layer by irradiation with radiation.
( 6 )
The fixing of the water-soluble resin to the biochemical instrument is mainly carried out by covalently bonding the first functional group and the second functional group ( 2 ) to ( 5 ). The manufacturing method of the biochemical instrument in any one .

  ADVANTAGE OF THE INVENTION According to this invention, the biochemical instrument which can reduce adsorption | suction of a biological substance and its manufacturing method can be provided.

  In addition, the biochemical device is immersed in a water-soluble resin having a first functional group in the side chain to form a coating layer on the surface, and the coating layer formed after the coating is cured by light irradiation or radiation irradiation is particularly useful for living organisms. It is possible to provide a biochemical instrument that has a small amount of adsorption of a derived substance and is excellent in heat resistance and organic solvent resistance.

  Hereinafter, the present invention will be described in detail.

  The method for producing a biochemical instrument of the present invention includes a first step of immersing in a water-soluble resin having a functional group in a side chain to form a coating layer on the surface of the biochemical instrument, and curing the coating layer In the second step, the water-soluble resin is fixed to the surface of the biochemical instrument.

  The water-soluble resin having the first functional group in the side chain preferably contains a photosensitive reactive group, a radiation reactive reactive group, and a heat sensitive reactive group. Among these, it is particularly preferable to contain a photosensitive reactive group. Thereby, the water-soluble resin having the first functional group in the side chain can be cross-linked efficiently in a short time with a simple production facility.

  Examples of the first functional group include a functional group containing a nitrogen atom, a functional group containing a sulfur atom, a functional group containing a bromine atom, a functional group containing a chlorine atom, or a functional group containing neither of these atoms. Is mentioned. Among these, a functional group containing a nitrogen atom is preferable.

  Specific examples include a functional group containing an azide group, a functional group containing a diazo group, and a functional group containing a diazide group. Among these, a functional group containing an azide group is preferable. Thereby, it can be made to react with the wavelength of practical 300-500 nm, and the formability of a film | membrane can be improved with the further outstanding resolution.

  Examples of the water-soluble resin constituting the water-soluble resin having the first functional group in the side chain include saponified polyvinyl acetate, polyvinyl pyrrolidone, polyethylene glycol, polyacrylamide, polymethacrylamide, polyhydroxyethyl methacrylate, poly Pentaerythritol triacrylate, polypentaerythritol tetraacrylate, polydiethylene glycol diacrylate, and copolymers of monomers constituting them, and copolymers of 2-methacryloyloxyethyl phosphorylcholine and other monomers (for example, butyl methacrylate, etc.) Etc. Among these, one or more selected from saponified products of polyvinyl acetate, polyvinyl pyrrolidone, and polyethylene glycol are preferable. Thereby, the reduction effect of the adsorption amount of a biological substance can be improved.

  Here, the polyvinyl acetate fight is a polyvinyl alcohol or a copolymer of vinyl alcohol and another compound. Further, vinyl alcohol and modified vinyl acetate saponified products such as hydrophilic group modification, hydrophobic group modification, anion modification, cation modification, amide group modification or reactive group modification such as acetoacetyl group are included.

  In addition, water-soluble resin means what can melt | dissolve 1.0g or more with respect to 100g of 25 degreeC water here.

  The average degree of polymerization of the water-soluble resin is not particularly limited, but is preferably 100 to 10,000, and particularly preferably 200 to 5,000. If the average degree of polymerization is less than the lower limit, it may be difficult to uniformly form a film on the surface of the biochemical instrument, and if the upper limit is exceeded, the side chain has the first functional group. In some cases, the viscosity of the water-soluble resin increases and the workability decreases.

  Further, when the saponified product of polyvinyl acetate is used, the saponification degree of the saponified product of polyvinyl acetate is not particularly limited, but is preferably 20 to 100 mol%, particularly preferably 50 to 95 mol% of the whole polyvinyl acetate. When the degree of saponification of the polyvinyl acetate is within the above range, the effect of reducing the amount of adsorption of the biological substance is particularly excellent.

As the water-soluble resin having the first functional group in the side chain, for example, a resin represented by the following formula (Ia ) is preferable. Thereby, a uniform film can be formed at a practical wavelength of 300 to 500 nm, and the effect of reducing the adsorption of a biological substance can be particularly improved.

R represented by the formula (Ia ) of the water-soluble resin is not particularly limited as long as it is an alkyl group having a carbonyl and an amine, but for example, those represented by the following formula (II) are preferable. Thereby, the synthesis of the first functional group can be easily performed.

  When the biochemical instrument is immersed in the water-soluble resin having the first functional group in the side chain, the water-soluble resin having the first functional group in the side chain is preferably immersed in a solvent, The solvent used in this case may be water or a mixture of water and an organic solvent in order to increase solubility.

For example, when the water-soluble resin represented by the formula (II) is used, 5 to 40% by volume
By using the aqueous alcohol solution as the solvent, the solubility of the water-soluble resin is increased, and a uniform coating layer can be formed.

  The concentration of the water-soluble resin to be dissolved is preferably 0.01 to 20% by weight, particularly preferably 0.1 to 5% by weight.

  If the concentration of the water-soluble resin is less than the lower limit value, a uniform coating layer cannot be obtained, and if the upper limit value is exceeded, the coating layer becomes too thick, and both have a sufficient effect of reducing the adsorption of biological substances. I can't get it.

  The thickness of the coating layer capable of obtaining the effect of reducing the adsorption of biological substances is preferably 2 to 3,000 nm, and more preferably 5 to 2,000 nm.

  As described above, a coating layer having a thickness suitable for the effect of reducing the adsorption of a biological substance can be obtained by forming a coating layer on the surface of the biochemical instrument in advance.

  It is preferable that the second functional group is formed on the surface of the biochemical instrument beforehand by corona treatment, plasma treatment or the like before immersion, and it is particularly preferable that a hydroxyl group or a carboxyl group is introduced by oxygen plasma treatment. .

  By introducing these polar groups, the affinity between the biochemical instrument surface and the water-soluble resin is improved, and as a result, a uniform coating layer is obtained. Furthermore, a coating layer that is firmly fixed to the biochemical instrument surface by the covalent bond between the formed second functional group and the first functional group is obtained, and the heat resistance and solvent resistance are improved. Excellent.

  Next, the second step, that is, the step of curing the formed coating layer will be described.

  The coating layer formed in the first step is cured in the second step, thereby obtaining water resistance and providing basic performance that can be used as a biochemical instrument. Furthermore, a surface having reduced adsorption of a biological substance having heat resistance and organic solvent resistance, which is a feature of the present invention, is obtained.

  Specifically, when the water-soluble resin itself is cross-linked by curing to become water-insoluble, and when the second functional group is introduced in advance on the surface of the biochemical instrument, the first functional group and The covalent bond with the second functional group can further improve the surface resistance to chemical and physical stimuli. In addition to the covalent bond between the first functional group and the second functional group, a covalent bond such as a carbon-hydrogen bond or a carbon-carbon bond on the surface of the biochemical instrument may be present. Good.

  The curing method is not particularly limited as long as the first functional group in the side chain of the water-soluble resin can react. For example, it is a photosensitive group such as a diazo group, an azide group, or a simmonyl group. It can be cured by light irradiation, and when it has a vinyl group, it can be cured by radiation.

A light source in the case of curing by light irradiation is not particularly limited, illumination can also be used 5.0 mW / cm ² of about ultra-high pressure mercury lamp or 0.1 mW / cm ² about UV lamps. Curing by light irradiation can be controlled by illuminance and irradiation time, so when using a light source with low illuminance, the irradiation time can be lengthened, and when a highly reactive photosensitive group is selected, it is cured under a fluorescent lamp. It is also possible.

For example, when using an extra-high pressure mercury lamp with 5.0 mW / cm ² irradiation 1 to 10 seconds, when using a UV lamp 0.1 mW / cm ² be sufficiently cured by irradiation of 3 to 10 minutes I can do it.

  Next, a biochemical instrument will be described.

  The biochemical instrument is made of the resin material. In addition to making the biochemical instrument a disposable type, the resin material can be easily molded into various shapes.

  Examples of the resin material include polypropylene resins, polyethylene resins, polyolefin resins such as ethylene-propylene copolymers, polystyrene resins such as polystyrene and acrylonitrile-butadiene-styrene resins, polycarbonate resins, polyethylene terephthalate resins, and polymethyl methacrylate. Methacrylic resin such as resin, vinyl chloride resin, polybutylene terephthalate resin, polyarylate resin, polysulfone resin, polyethersulfone resin, polyetheretherketone resin, polyetherimide resin, fluorine resin such as polytetrafluoroethylene, Examples thereof include acrylic resins such as polymethylpentene resin and polyacrylonitrile, and fiber-based resins such as propionate resin. Among these, a polypropylene resin is particularly preferable in terms of moldability, transparency, resistance to chemical and physical irritation required for biochemical instruments.

  The weight average molecular weight of the resin material is not particularly limited, but is preferably 10,000 to 500,000, particularly preferably 20,000 to 100,000. When the weight average molecular weight is within the above range, the moldability of the biochemical instrument is excellent, and the molded product is excellent in water resistance.

  The weight average molecular weight is, for example, G.M. P. C. It can obtain | require in conversion of styrene using.

  For the purpose of improving moldability and weather resistance, additives such as hydrocarbon-based, fatty acid amide-based lubricants, phenol-based and amine-based antioxidants are added to the resin material as long as the object of the present invention is not impaired. can do.

  When the biochemical instrument is manufactured from the resin material, the biochemical instrument can be manufactured by, for example, injection molding, blow molding, or injection blow molding.

  Examples of the biochemical instrument include liquid manipulation instruments such as pipettes and dispenser chips used for biochemical research, and containers such as centrifuge tubes, cryopreservation tubes, tubes, and multiwell plates. Among these, the contact time with a biological substance is relatively long, and it is used as tubes (specifically, blood collection containers or test tubes and sample containers used for protein analysis) used for sample preparation in various analyses. It is preferred that This improves the accuracy and sensitivity of the analysis results.

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and a comparative example, this invention is not limited to this.
Example 1
Polypropylene resin (manufactured by Sumitomo Noblen Co., WP708) is used as a resin material, and injection molding (molding machine: manufactured by Nissei Plastic Industries 60t, cylinder temperature: 225 ° C-220 ° C-195 ° C-185 ° C, injection rate: 25%- 20% -15%, injection pressure: 40% -30% -25%, mold cooling: 30 ° C.). The obtained tube was subjected to plasma treatment (oxygen plasma for 5 minutes) using a plasma treatment apparatus (SERIES7000 manufactured by BRANSON / IPC) to form second functional groups on the surface of the tube.

  In addition, the shape of the obtained tube was a V bottom tube having a height of 39 mm, an outer diameter of 10 mm, and a capacity of 1.5 mL.

  Next, polyvinyl alcohol having an azide group in the side chain as a water-soluble resin having a first functional group in the side chain (AWP manufactured by Toyo Gosei Kogyo Co., Ltd., average polymerization degree of water-soluble resin 1,800, first functional group Was dissolved in a 20 vol% ethanol aqueous solution in a glass container shielded from light with an aluminum foil to prepare a 1.0 wt% solution.

The tube on which the second functional group is formed is dipped in a glass container shielded with aluminum foil for 1 minute, then taken out, dried at 40 ° C. for 60 minutes, and then subjected to UV light with a UV lamp at 0.1 mW. Irradiated with / cm ² × 5 minutes to cure the water-soluble resin having the first functional group on the side chain and then washed with pure water to obtain the biochemical instrument (tube) of the present invention.

On the surface of the obtained tube, a layer formed of a water-soluble resin having the first functional group in the side chain was formed with a thickness of 250 nm.
(Example 2)
Except for irradiating UV light with 5.0 mW / cm ² × 3 seconds with an ultrahigh pressure mercury lamp instead of the UV lamp to cure the water-soluble resin having the first functional group on the side chain, the same as in Example 1. did.

On the surface of the obtained tube, a layer formed of a water-soluble resin having the first functional group in the side chain was formed with a thickness of 250 nm.
(Example 3)
The same procedure as in Example 1 was performed except that the water-soluble resin having the first functional group in the side chain was cured by irradiation (γ-ray 5 kGy) instead of the UV lamp.

On the surface of the obtained tube, a layer formed of a water-soluble resin having the first functional group in the side chain was formed with a thickness of 250 nm.
(Example 4)
The same procedure as in Example 1 was performed except that the tube was not previously plasma-treated and the second functional group was not formed.

On the surface of the obtained tube, a layer formed of a water-soluble resin having the first functional group in the side chain was formed with a thickness of 180 nm.
(Example 5)
Methyl pentene (TPX) resin (manufactured by Mitsui Petrochemical Co., Ltd., RT-31) was used as the resin material, and the same procedure as in Example 1 was performed except that the injection molding conditions were as follows.

  Injection molding machine: Nissei Plastic Industries 60t, cylinder temperature: 300 ° C-280 ° C-265 ° C-255 ° C, injection speed: 40% -30% -10%, injection pressure: 60% -30% -25% Mold cooling: performed at 50 ° C.

On the surface of the obtained tube, a layer formed of a water-soluble resin having the first functional group in the side chain was formed with a thickness of 250 nm.
(Comparative Example 1)
The tube with the second functional group formed was immersed in a glass container shielded with aluminum foil for 1 minute, then taken out without drying, and simultaneously irradiated with UV light, as in Example 1. did.

On the surface of the obtained tube, a layer formed of a water-soluble resin having the first functional group in the side chain was formed in a thickness range of 300 nm to 5000 nm.
(Comparative Example 2)
Polyvinyl alcohol having no functional group in the side chain as a water-soluble resin: Examples except that an average polymerization degree of about 1,500 and a saponification degree of 86 to 90 mol% (manufactured by Wako Pure Chemical Industries, Ltd., 160-03055) were used Same as 1.

The following evaluation was performed about the obtained container. The evaluation items are shown together with the contents. The obtained results are shown in Table 1.
1. Comparison of Adsorbability of Biological Substances 1.1 Bovine Serum Immunoglobulin Adsorbability The adsorptivity of bovine serum immunoglobulin was evaluated as follows. Bovine serum immunoglobulin (manufactured by Bovine gamma globulin standard 23210 PIERCE) was labeled with iodine ( ¹²⁵ I) and diluted with phosphate buffer (pH 7.4) to a concentration of 1.0E ⁻⁷ g / mL. It dispensed into the container obtained by the comparative example, and left still at 37 degreeC for 1 hour. Thereafter, washing was repeated 3 times with 0.05 vol% Tween20-containing phosphate buffer, and the container body was measured with a γ-ray counter.

The weight of iodine ( ¹²⁵ I) -labeled bovine serum immunoglobulin remaining in the container body was determined from a separately prepared calibration curve, and the adsorption rate at each solution concentration was calculated.

1.2 Bovine serum albumin adsorptivity The adsorptivity of bovine serum albumin was evaluated as follows. A solution obtained by diluting bovine serum albumin (manufactured by 23209 PIERCE) to 0.5 μg / mL was dispensed at 1.0 mL each into the containers obtained in each Example and Comparative Example, and incubated at 37 ° C. for 1 hour. Washed 3 times with a phosphate buffer pH 7.4 containing 0.05% by volume tween20.

  Next, as a blocking, a phosphate buffer solution pH 7.4 containing 3.0% by weight skimmed milk was dispensed in 1.5 mL portions, incubated at 37 ° C. for 1 hour, and then phosphate buffer solution pH 7.4 containing 0.05% by volume tween20. And washed 3 times. Next, a solution obtained by diluting peroxidase-labeled anti-bovine serum albumin antibody (55285 CAPPEL) to 1.0 μg / mL with phosphate buffer pH 7.4 was dispensed at 1.0 mL in each container and incubated at room temperature for 30 minutes. After washing with 0.05 volume% tween 20 phosphate buffer pH 7.4 three times, color was developed using a peroxidase color development kit (SUMILON ML-1120T manufactured by Sumitomo Bakelite Co., Ltd.) and a plate reader was used. The absorbance at 450/630 nm was measured.

  The weight of peroxidase-labeled anti-bovine serum albumin antibody = bovine serum albumin remaining in the container body was determined from a separately prepared calibration curve, and the adsorption rate was calculated.

1.3 Adsorbability of peroxidase-labeled avidin The adsorptivity of peroxidase-labeled avidin was evaluated as follows. A solution obtained by diluting peroxidase-labeled avidin (43-4423, manufactured by ZYMED) to 0.5 μg / mL was dispensed into each of the containers obtained in each Example and Comparative Example in an amount of 1.0 mL and allowed to stand at room temperature for 1 hour. Then, the plate was washed three times with a phosphate buffer solution pH 7.4 containing 0.05% tween 20, and developed using a peroxidase coloring kit (SUMILON ML-1120T manufactured by Sumitomo Bakelite Co., Ltd.), and then a plate reader was used. The absorbance at 450/630 nm was measured.

The weight of peroxidase-labeled avidin remaining in the container body was determined from a separately prepared calibration curve, and the adsorption rate was calculated.
2. Measurement of eluate The eluate was measured with a UV spectrophotometer.

Pure water was dispensed in 1.5 mL increments into the containers obtained in each Example and Comparative Example, and the dispensed pure water was recovered after incubation at 37 ° C. for 24 hours, and the UV spectrophotometer measured 220 nm to 300 nm. The presence or absence of absorption was confirmed.
3. Solvent resistance The solvent resistance was evaluated by the adsorption rate of bovine serum immunoglobulin G after the following treatment.

A 50 volume% aqueous solution of acetonitrile was dispensed in 1.5 mL aliquots into the containers obtained in each of the examples and comparative examples, left standing for 10 minutes, washed with pure water, and evaluated as described above (bovine serum immunoglobulin adsorptivity). Thus, the adsorptivity of bovine serum immunoglobulin G was evaluated.
4). Centrifugal strength Centrifugal strength was evaluated using a centrifuge.

Dispense 1.5 mL of pure water into the containers obtained in each Example and Comparative Example, and set in a centrifuge (CF16RX type Hitachi Multi-purpose Small Centrifuge Rotor # 11). After centrifuging for minutes, the container was visually checked for deformation and cracking.
5. Heat resistance The heat resistance was evaluated by the deformation of the container after the following treatment and the adsorption rate of bovine serum immunoglobulin G.

  Dispense 1.5 mL of pure water into the containers obtained in each Example and Comparative Example, leave it in a thermostatic bath at 95 ° C. for 10 minutes, discard the pure water, and visually check the container for deformation and cracking. did.

Further, the adsorptivity of bovine serum immunoglobulin G was evaluated by the method for evaluating the adsorptivity of bovine serum immunoglobulin.
6). Evaluation of cold resistance Cold resistance was evaluated by the deformation of the container after the following treatment and the adsorption rate of bovine serum immunoglobulin G.

  Dispense 1.5 mL of pure water into the containers obtained in each Example and Comparative Example, and leave it in a deep freezer at −80 ° C. for 24 hours, then discard the pure water, and visually observe deformation and cracking of the tube body. It was confirmed.

  Further, the adsorptivity of bovine serum immunoglobulin G was evaluated by the method for evaluating the adsorptivity of bovine serum immunoglobulin.

  As apparent from Table 1, it was shown that Examples 1 to 5 were able to reduce the adsorption amount of the biological substance. Thereby, it can use suitably for a blood collection container or a test tube and a sample container used for protein analysis.

  In addition, in Examples 1 to 3 and 5, the amount of the eluate was particularly small, which can prevent the direct influence on the sample and the influence on the measurement result due to the dissolution of the water-soluble resin. it can.

  Examples 1 to 5 were also excellent in solvent resistance and centrifugal strength.

  Moreover, Examples 1-5 were excellent also in heat resistance, and Examples 1-3 and 5 also had especially few adsorption amount of the biological origin substance after heat processing.

  Moreover, Examples 1-5 were excellent also in cold resistance, and the adsorption amount of the biological substance after freezing process was also especially small.

  As described above, Examples 1 to 5 have been shown to be suitable for containers for analyzing low-concentration biological substances because the adsorption rate of biological substances is low.

  The present invention is a surface modification method for reducing the amount of biological material adsorbed on the surface of a biochemical instrument, and in particular, biochemical research such as genetic engineering, protein engineering, and clinical laboratory science, and analysis of biological material. When applied to instruments such as pipettes, dispenser chips, storage containers, dilution containers used when performing, there is no loss due to adsorption of samples such as biological substances, and organic solvents can be used with high accuracy and Highly sensitive analysis can be performed.

Claims (6)

A first step of immersing the biochemical instrument in a water-soluble resin having a first functional group in the side chain to form a coating layer on the surface of the biochemical instrument;
A second step of curing the coating layer,
The water-soluble resin having the first functional group in the side chain is polyvinyl alcohol obtained by saponifying polyvinyl acetate, and the polyvinyl alcohol further includes a structural unit represented by the following formula (Ia ). A method for manufacturing a biochemical instrument.

The method for producing a biochemical instrument according to claim 1, wherein a second functional group is formed in advance on the surface of the biochemical instrument. The method for producing a biochemical instrument according to claim 1 or 2, wherein the biochemical instrument is made of resin. The method of manufacturing a biochemical instrument according to any one of claims 1 to 3, wherein in the second step, the coating layer is cured by light irradiation. The method for producing a biochemical instrument according to any one of claims 1 to 3, wherein in the second step, the coating layer is cured by irradiation with radiation. The fixing of the water-soluble resin to the biochemical instrument is mainly performed by covalently bonding the first functional group and the second functional group. The manufacturing method of the biochemical instrument as described in any one of.